中国中部地区间日疟原虫DBP II克隆表达及其鉴定
发布时间：2018-08-30 07:58
【摘要】：目的（1）克隆中国安徽流行区间日疟原虫达菲血型结合蛋白II区基因（Plasmodium vivax Duffy Binding Protein region II，PvDBPII）；（2）体外原核表达和鉴定重组PvDBPII蛋白。 方法（1）根据GenBank收录的间日疟原虫Sa1-1株PvDBPII基因（GI：156081788）序列设计引物，从镜检阳性的间日疟原虫感染血样本中抽提DNA为模板，，应用PCR技术从间日疟基因组DNA中扩增DBPII基因。PvDBPII PCR产物与pET-28a载体分别经Nco I和Hind III双酶切后连接克隆入原核表达载体pET28a(+)中，构建pET28a(+)-PvDBPII重组表达质粒，采用双酶切及测序方法鉴定重组质粒。（2）将重组质粒pET28a(+)-PvDBPII转化至大肠埃希菌BL21(DE3+)中，IPTG诱导表达带有His标签的重组蛋白，采用镍柱亲和层析纯化重组蛋白，采用SDS-PAGE电泳和Western Blotting分析鉴定表达产物。 结果（1）PCR体外扩增得到目的基因片段长约1.1kb。双酶切及测序结果显示目的基因己被正确地连接入重组质粒pET28a(+)中，其插入片段序列与Sa1-1株PvDBPII基因参考序列相比存在四个非同义突变位点（R319G、D384G、R390H和L424I）。（2）重组质粒转入大肠埃希菌后，诱导表达的重组蛋白分子量约为44kDa，且能被间日疟患者血浆特异性识别。在37℃，IPTG0.5mmol/L诱导表达4h可达到最大表达量，表达产物为不可溶包涵体。 结论成功克隆了中国安徽流行区间日疟原虫PvDBPII基因，表达出了重组PvDBPII蛋白，并优化了PvDBPII重组蛋白的原核表达条件，验证了其抗原性，为进一步研究基于PvDBPII的红内期间日疟疫苗提供了基础；
[Abstract]:Objective (1) to clone the gene (Plasmodium vivax Duffy Binding Protein region II,PvDBPII); (2 of II region of Tamiflu blood group binding protein of Plasmodium vivax in Anhui Province, China, and to express and identify the recombinant PvDBPII protein in vitro. Methods (1) primers were designed according to the PvDBPII gene (GI:156081788) sequence of Plasmodium vivax Sa1-1 strain included in GenBank, and DNA was extracted from infected blood samples of Plasmodium vivax positive by microscope as template. The DBPII gene, PvDBPII PCR product and pET-28a vector were amplified from vivax malaria genomic DNA by PCR technique and cloned into the prokaryotic expression vector pET28a () by double enzyme digestion of Nco I and Hind III, respectively. The recombinant expression plasmid of pET28a () -PvDBPII was constructed. The recombinant plasmid was identified by double enzyme digestion and sequencing. (2) the recombinant plasmid pET28a () -PvDBPII was transformed into Escherichia coli BL21 (DE3) to induce the expression of recombinant protein with His label, and the recombinant protein was purified by nickel column affinity chromatography. The expressed products were identified by SDS-PAGE electrophoresis and Western Blotting analysis. Results (1) the length of the target gene fragment was about 1.1 kb by PCR amplification in vitro. The results of double enzyme digestion and sequencing showed that the target gene had been correctly ligated into the recombinant plasmid pET28a (). Compared with the reference sequence of the PvDBPII gene of Sa1-1 strain, there were four non-synonymous mutational sites (R319GN, D384GN, R390H and L424I). (2) in the recombinant plasmid transformed into Escherichia coli. The molecular weight of the recombinant protein induced was about 44 kDa and could be specifically recognized by the plasma of vivax malaria patients. The expression of IPTG 0.5 mmol / L at 37 鈩
本文编号：2212465
[Abstract]:Objective (1) to clone the gene (Plasmodium vivax Duffy Binding Protein region II,PvDBPII); (2 of II region of Tamiflu blood group binding protein of Plasmodium vivax in Anhui Province, China, and to express and identify the recombinant PvDBPII protein in vitro. Methods (1) primers were designed according to the PvDBPII gene (GI:156081788) sequence of Plasmodium vivax Sa1-1 strain included in GenBank, and DNA was extracted from infected blood samples of Plasmodium vivax positive by microscope as template. The DBPII gene, PvDBPII PCR product and pET-28a vector were amplified from vivax malaria genomic DNA by PCR technique and cloned into the prokaryotic expression vector pET28a () by double enzyme digestion of Nco I and Hind III, respectively. The recombinant expression plasmid of pET28a () -PvDBPII was constructed. The recombinant plasmid was identified by double enzyme digestion and sequencing. (2) the recombinant plasmid pET28a () -PvDBPII was transformed into Escherichia coli BL21 (DE3) to induce the expression of recombinant protein with His label, and the recombinant protein was purified by nickel column affinity chromatography. The expressed products were identified by SDS-PAGE electrophoresis and Western Blotting analysis. Results (1) the length of the target gene fragment was about 1.1 kb by PCR amplification in vitro. The results of double enzyme digestion and sequencing showed that the target gene had been correctly ligated into the recombinant plasmid pET28a (). Compared with the reference sequence of the PvDBPII gene of Sa1-1 strain, there were four non-synonymous mutational sites (R319GN, D384GN, R390H and L424I). (2) in the recombinant plasmid transformed into Escherichia coli. The molecular weight of the recombinant protein induced was about 44 kDa and could be specifically recognized by the plasma of vivax malaria patients. The expression of IPTG 0.5 mmol / L at 37 鈩
本文编号：2212465
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